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1.
Soft Matter ; 17(31): 7260-7267, 2021 Aug 11.
Article in English | MEDLINE | ID: mdl-34337643

ABSTRACT

Fluorosurfactants have expanded the landscape of high-value biochemical assays in microfluidic droplets, but little is known about how the spatial geometries and polarity of the head group contribute to the performance of fluorosurfactants. To decouple this, we design, synthesize, and characterize two linear and two dendritic glycerol- or tris-based surfactants with a common perfluoropolyether tail. To reveal the influence of spatial geometry, we choose inter-droplet cargo transport as a stringent test case. Using surfactants with linear di- and triglycerol, we show that the inter-droplet cargo transport is minimal compared with their dendritic counterparts. When we encapsulated a less-leaky sodium fluorescent dye into the droplets, quantitatively, we find that the mean fluorescence intensity of the PFPE-dTG stabilized PBS-only droplets after 72 h was ∼3 times that of the signal detected in PBS-only droplets stabilized by PFPE-lTG. We also demonstrate that the post-functionalization of PFPE-lTG having a linear geometry and four hydroxy groups enables the 'from-Droplet' fishing of the biotin-streptavidin protein complex without the trade-off between fishing efficiency and droplet stability. Thus, our approach to design user-friendly surfactants reveals the aspects of spatial geometry and facile tunability of the polar head groups that have not been captured or exploited before.


Subject(s)
Microfluidic Analytical Techniques , Microfluidics , Fluorescent Dyes , Surface-Active Agents
2.
Lab Chip ; 20(8): 1513-1520, 2020 04 21.
Article in English | MEDLINE | ID: mdl-32242586

ABSTRACT

Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.


Subject(s)
Lab-On-A-Chip Devices , Microfluidics , Cytokines , Killer Cells, Natural
3.
Nature ; 578(7796): 593-599, 2020 02.
Article in English | MEDLINE | ID: mdl-32051591

ABSTRACT

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.


Subject(s)
Astrocytes/pathology , Central Nervous System/pathology , Inflammation/pathology , MafG Transcription Factor/genetics , Repressor Proteins/genetics , Animals , Antioxidants/metabolism , Astrocytes/metabolism , Central Nervous System/metabolism , DNA Methylation , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Inflammation/genetics , Male , Methionine Adenosyltransferase/genetics , Mice , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , NF-E2-Related Factor 2/genetics , Sequence Analysis, RNA , Signal Transduction , Transcription, Genetic
4.
RSC Adv ; 10(45): 27006-27013, 2020 Jul 15.
Article in English | MEDLINE | ID: mdl-35515810

ABSTRACT

Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.

5.
Chem Commun (Camb) ; 56(4): 647-650, 2020 Jan 14.
Article in English | MEDLINE | ID: mdl-31840153

ABSTRACT

Here, we demonstrate use of a Mg2+-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.


Subject(s)
DNA, Catalytic/metabolism , Nucleic Acids/metabolism , Acrylic Resins/chemistry , Acrylic Resins/metabolism , Gels/chemistry , Gels/metabolism , Immobilized Nucleic Acids/chemistry , Immobilized Nucleic Acids/metabolism , Magnesium/chemistry , Magnesium/metabolism , Nucleic Acid Amplification Techniques , Nucleic Acids/chemistry , Particle Size , Surface Properties
6.
Lab Chip ; 19(24): 4064-4070, 2019 12 21.
Article in English | MEDLINE | ID: mdl-31690904

ABSTRACT

We report an additive-free method to lyse bacteria and extract nucleic acids and protein using a traveling surface acoustic wave (TSAW) coupled to a microfluidic device. We characterize the effects of the TSAW on E. coli by measuring the viability of cells exposed to the sound waves and find that about 90% are dead. In addition, we measure the protein and nucleic acids released from the cells and show that we recover about 20% of the total material. The lysis method should work for all types of bacteria. These results demonstrate the feasibility of using TSAW to lyse bacteria in a manner that is independent of the type of bacteria.


Subject(s)
Escherichia coli/chemistry , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , Sound , Equipment Design , Escherichia coli/cytology
7.
J Ind Microbiol Biotechnol ; 46(11): 1603-1610, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31375945

ABSTRACT

The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.


Subject(s)
Cellulase/metabolism , Microfluidics , Trichoderma/metabolism , Gene Library , Trichoderma/genetics
8.
Chem Commun (Camb) ; 55(51): 7358-7361, 2019 Jun 20.
Article in English | MEDLINE | ID: mdl-31172143

ABSTRACT

Here, we describe a simple mix-and-read method for the detection of specific bacterial strains that uses a DNAzyme and a molecular beacon to generate a signal. We have greatly improved upon the previously described DNAzyme-based bacteria detection method by eliminating a tedious preparation step while maintaining detection sensitivity.


Subject(s)
Cell Extracts/analysis , DNA, Bacterial/analysis , DNA, Catalytic/metabolism , DNA, Single-Stranded/analysis , Escherichia coli/classification , Biosensing Techniques , Fluorescent Dyes/chemistry , Limit of Detection
9.
Biomicrofluidics ; 11(1): 014114, 2017 Jan.
Article in English | MEDLINE | ID: mdl-28344725

ABSTRACT

Active manipulation of droplets is crucial in droplet microfluidics. However, droplet polydispersity decreases the accuracy of active manipulation. We develop a microfluidic "droplet filter" that accurately separates droplets by size. The droplet filter has a sharp size cutoff and is capable of distinguishing droplets differing in volume by 20%. A simple model explains the behavior of the droplets as they pass through the filter. We show application of the filter in improving dielectric sorting efficiency.

10.
Nat Protoc ; 8(5): 870-91, 2013 May.
Article in English | MEDLINE | ID: mdl-23558786

ABSTRACT

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.


Subject(s)
Flow Cytometry/methods , Single-Cell Analysis/methods , Animals , Antibodies/analysis , Fluorescent Dyes , Hybridomas/metabolism , Mice , Microfluidic Analytical Techniques/instrumentation , Microfluidics/methods
11.
J Am Chem Soc ; 134(10): 4983-9, 2012 Mar 14.
Article in English | MEDLINE | ID: mdl-22356466

ABSTRACT

Micrometer-sized hydrogel particles that contain living cells can be fabricated with exquisite control through the use of droplet-based microfluidics and bioinert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). However, in existing techniques, the microgel gelation is often achieved through harmful reactions with free radicals. This is detrimental for the viability of the encapsulated cells. To overcome this limitation, we present a technique that combines droplet microfluidic templating with bio-orthogonal thiol-ene click reactions to fabricate monodisperse, cell-laden microgel particles. The gelation of these microgels is achieved via the nucleophilic Michael addition of dithiolated PEG macro-cross-linkers to acrylated hPG building blocks and does not require any initiator. We systematically vary the microgel properties through the use of PEG linkers with different molecular weights along with different concentrations of macromonomers to investigate the influence of these parameters on the viability and proliferation of encapsulated yeast cells. We also demonstrate the encapsulation of mammalian cells including fibroblasts and lymphoblasts.


Subject(s)
Gels , Microfluidics/methods , Animals , Cells, Cultured , Kinetics , Mammals , Saccharomyces cerevisiae/chemistry
12.
Lab Chip ; 12(12): 2146-55, 2012 Jun 21.
Article in English | MEDLINE | ID: mdl-22318506

ABSTRACT

Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10(8) samples to be screened in one day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.


Subject(s)
High-Throughput Screening Assays , Microfluidics , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antibodies/immunology , Antibodies/isolation & purification , Bacteria/drug effects , DNA/analysis , Enzymes/chemistry , Enzymes/metabolism , Humans , Polymerase Chain Reaction , Viral Plaque Assay
13.
Biomaterials ; 32(5): 1311-6, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21047679

ABSTRACT

We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 µm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.


Subject(s)
Biocompatible Materials/adverse effects , Biocompatible Materials/chemistry , Glycerol/chemistry , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , Polymers/chemistry , Glycerol/adverse effects , Microspheres , Nanogels , Nanoparticles/adverse effects , Nanoparticles/chemistry , Polyethylene Glycols/adverse effects , Polyethyleneimine/adverse effects , Polymers/adverse effects , Yeasts/cytology , Yeasts/drug effects
14.
Genes Dev ; 16(6): 707-19, 2002 Mar 15.
Article in English | MEDLINE | ID: mdl-11914276

ABSTRACT

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass approximately 5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability--a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu.


Subject(s)
Genome, Fungal , Saccharomyces cerevisiae/metabolism , Algorithms , Cell Nucleus/metabolism , Chromosomes/metabolism , Cytoplasm/metabolism , Databases as Topic , Epitopes , Microscopy, Fluorescence , Mitochondria/metabolism , Models, Genetic , Mutagenesis , Phenotype , Software
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